Recent studies have shown that RNAs are invariably bound to and often modified by RNA-binding proteins (RBPs). Thus, it is no surprise that RBPs have been found to play key roles in regulating many aspects of coding and non-coding RNA biology, including RNA processing, nuclear export, cellular transport, function, localization, and stability. These efforts are carried out by >1,500 unique RBPs that utilize a variety of RNA- binding domains to achieve oftentimes specific and high affinity interactions with target transcripts; however, non-canonical RBPs have also been identified. Disruption of this complex network of RNA-protein interactions (RPIs) has been implicated in a number of human diseases. Thus, the targeting of RBPs and RPIs has arisen as a new frontier in RNA-targeted drug discovery; however, very few interactions have been validated to support a pipeline of targets for these efforts. While the advent of sequencing and quantitative mass spectrometry has dramatically enhanced our ability to globally profile these interactions, experimental validation of these data sets remains a challenge. Using chemical biology- and bioorthogonal chemistry-based strategies, we have developed an innovative new assay for the live-cell detection of RPIs, RNA interaction with Protein-mediated Complementation Assay, or RiPCA. Through this approach, we have detected the interaction of pre-miRNAs with RBPs, in addition to inhibition with small molecules. Moreover, to provide evidence for the potential of our technology in validating new RPIs, we used RiPCA to confirm the interaction of a pre-miRNA with a novel RBP discovered via proteomics. In total, these data provide encouraging proof-of- concept for this emerging technology; yet, many key questions and challenges still remain to ensure that RiPCA is a rigorous and unbiased approach for the detection of RPIs in distinct cellular organelles. In Specific Aim 1, we will further develop RiPCA for organelle-specific detection to ensure its accuracy. In Specific Aim 2, we will investigate the potential of the assay by profiling additional RPIs from various RNA and RBP families. Finally, in Specific Aim 3, we will explore its adaptability toward high-throughput experimentation for validation of large-scale CLIP or proteomics data sets, or screening to identify cell-active small molecule inhibitors of RPIs. Upon completion of the proposed research, our goal is to produce a robust and user-friendly technology for the rapid validation and study of cellular RPIs to enable biomedical research.